The present invention is directed to methods for enhancing sequence specificity, binding affinity and solubility of peptide nucleic acids (PNAs) in which naturally-occurring nucleobases or non-naturally-occurring nucleobases are covalently bound to a polyamide backbone. The PNAs of the present invention comprise at least one C1-C8 alkylamine side chain resulting in enhanced solubility, binding affinity to nucleic acids and sequence specificity as well as other beneficial qualities. In certain aspects, the present invention is directed to histidine-containing peptide nucleic acids and to synthetic intermediates employed in preparing such compounds.
The function of a gene starts by transcription of its information to a messenger RNA (mRNA). By interacting with the ribosomal complex, mRNA directs synthesis of the protein. This protein synthesis process is known as translation. Translation requires the presence of various cofactors, building blocks, amino acids and transfer RNAs (tRNAs), all of which are present in normal cells.
Most conventional drugs exert their effect by interacting with and modulating one or more targeted endogenous proteins, e.g., enzymes. Typically, however, such drugs are not specific for targeted proteins but interact with other proteins as well. Thus, use of a relatively large dose of drug is necessary to effectively modulate the action of a particular protein. If the modulation of a protein activity could be achieved by interaction with or inactivation of mRNA, a dramatic reduction in the amount of drug necessary, and the side-effects of the drug, could be achieved. Further reductions in the amount of drug necessary and the side-effects could be obtained if such interaction is site-specific. Since a functioning gene continually produces mRNA, it would be even more advantageous if gene transcription could be arrested in its entirety. Oligonucleotides and their analogs have been developed and used as diagnostics, therapeutics and research reagents. One example of a modification to oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and 2xe2x80x2-O-methyl ribose sugar moieties. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotide analogs.
In the art, there are several known nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase. One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (Tm). This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is that Tm is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNAs have increased specificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8 to 20xc2x0 C. drop in the Tm relative to the DNA/DNA duplex. This decrease in Tm is not observed with the corresponding DNA/DNA duplex mismatch (Egholm et al., Nature 1993, 365, 566).
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5xe2x80x2 to 3xe2x80x2 orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5xe2x80x2 end of the DNA or RNA and amino end of the PNA is directed towards the 3xe2x80x2 end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are just the reverse with respect to the 5xe2x80x2-3xe2x80x2 direction of the DNA or RNA.
PNAs bind to both single stranded DNA and double stranded DNA. As noted above, in binding to double stranded DNA it has been observed that two strands of PNA can bind to the DNA. While PNA/DNA duplexes are stable in the antiparallel configuration, it was previously believed that the parallel orientation is preferred for (PNA)2/DNA.
The binding of two single stranded pyrimidine PNAs to a double stranded DNA has been shown to take place via strand displacement, rather than conventional triple helix formation as observed with triplexing oligonucleotides. When a PNA strand invades double stranded DNA, one strand of the DNA is displaced and forms a loop on the side of the PNA2/DNA complex area. The other strand of the DNA is locked up in the (PNA)2/DNA triplex structure. The loop area (alternately referenced as a D loop) being single stranded, is susceptible to cleavage by enzymes that can cleave single stranded DNA.
A further advantage of PNAs, compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
These properties make PNAs useful in several aapplications. Since PNAs have stronger binding and greater specificity than oligonucleotides, they are used as probes in cloning, blotting procedures, and in applications such as fluorescence in situ hybridization (FISH). Homopyrimidine PNAs are used for strand displacement in homopurine targets. The restriction sites that overlap with or are adjacent to the D-loop are not cleaved by restriction enzymes. Also, the local triplex inhibits gene transcription. Thus in binding of PNAs to specific restriction sites within a DNA fragment, cleavage at those sites can be inhibited. Advantage can be taken of this in cloning and subcloning procedures. Labeled PNAs are also used to directly map DNA molecules. In effecting this, PNA molecules having a fluorescent label are hybridized to complementary sequences in duplex DNA using strand invasion.
PNAs have further been used to detect point mutations in PCR-based assays (PCR clamping). PCR clamping uses PNA to detect point mutations in a PCR-based assay, e.g., the distinction between a common wild type allele and a mutant allele, in a segment of DNA under investigation. A PNA oligomer complementary to the wild type sequence is synthesized. The PCR reaction mixture contains this PNA and two DNA primers, one of which is complementary to the mutant sequence. The wild type PNA oligomer and the DNA primer compete for hybridization to the target. Hybridization of the DNA primer and subsequent amplification will only occur if the target is a mutant allele. With this method, one can determine the presence and exact identity of a mutant.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind to complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. For many applications, the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express their activity.
PCT/EP/01219 describes novel PNAs which bind to complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append groups to these PNAs which will modulate their activity, modify their membrane permiability or increase their cellular uptake property. One method for increasing amount of cellular uptake property of PNAs is to attach a lipophilic group. United States application Ser. No. 117,363, filed Sep. 3, 1993, describes several alkylamino functionalities and their use in the attachment of such pendant groups to oligonucleosides.
U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, and its corresponding published PCT application WO 94/06815, describe other novel amine-containing compounds and their incorporation into oligonucleotides for, inter alia, the purposes of enhancing cellular uptake, increasing lipophilicity, causing greater cellular retention and increasing the distribution of the compound within the cell.
U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, describes nucleosides and oligonucleosides derivatized to include a thiolalkyl functionality, through which pendant groups are attached.
Peptide nucleic acids may contain purine or pyrimidine nucleobases. However, previous PNAs having a high purine nucleobase content exhibit decreased solubility at physiological pH. PNAs of the present invention overcome this problem.
Despite recent advances, there remains a need for a stable compound that enhances or modulates binding to nucleic acids, stabilizes the hybridized complexes and increases the aqueous solubility.
The present invention provides methods for enhancing the solubility, sequence specificity and binding affinity of peptide nucleic acids (PNAs), for complementary DNA or RNA, by incorporation C1-C8 akylamine side chain into PNAs.
The peptide nucleic acids (PNAs) of the invention generally comprise ligands linked to a polyamide backbone. Representative ligands include the four major naturally-occurring DNA nucleobases (i.e. thymine, cytosine, adenine and guanine), other naturally-occurring nucleobases (e.g. inosine, uracil, 5-methylcytosine, thiouracil or 2,6-diaminopurine) or artificial bases (e.g. bromothymine, azaadenines or azaguanines) attached to a polyamide backbone through a suitable linker.
In one aspect, the present invention provides a method for enhancing the DNA or RNA sequence specificity of a peptide nucleic acid by incorporating C1-C8 akylamine side chain in said peptide nucleic acid having formula (I): 
wherein:
each L is independently selected from a group consisting of naturally-occurring nucleobases and non-naturally-occurring nucleobases;
each R7xe2x80x2 is independently hydrogen or C1-C8 alkylamine, provided that at least one R7xe2x80x2 is C1-8 alkylamine;
Rh is OH, NH2 or NHLysNH2;
Ri is H, COCH3 or t-butoxycarbonyl; and
n is an integer from 1 to 30.
Preferably, at least one R7xe2x80x2 is C3-C6 alkylamine. More preferably, at least one R7xe2x80x2 is C4-C5 alkylamine. Even more preferably, at least one R7xe2x80x2 is butylamine. Still even more preferably, substantially all of the R7xe2x80x2 are butylamine.
Preferably, the carbon atom to which substituent R7xe2x80x2 are attached is stereochemically enriched. Hereinafter, xe2x80x9cstereochemically enrichedxe2x80x9d means that one stereoisomer is present more than the other stereoisomer in a sufficient amount as to provide a beneficial effect. Preferably, one stereoisomer is present by more than 50%. More preferably, one stereoisomer is present by more than 80%. Even more preferably, one steroisomer is present by more than 90%. Still more preferably, one stereoisomer is present by more than 95%. Even more preferably, one stereoisomer is present by more than 99%. Still even more preferably, one stereoisomer is present in substantially quantitatively. Preferably, the stereochemical enrichment is of R configuration.
Preferably, the peptide nucleic acid is derived from an amino acid. More preferably, the peptide nucleic acid is derived from D-lysine.
The present invention also provides a method for enhancing the DNA or RNA binding affinity of a peptide nucleic acid by incorporating C1-C8 alkylamine side chain in the peptide nucleic acid having formula (I).
The present invention also provides a method for enhancing the solubility of a peptide nucleic acid by incorporating C1-C8 alkylamine side chain in the peptide nucleic acid having formula (I).
The PNAs of the invention are synthesized by adaptation of standard peptide synthesis procedures, either in solution or on a solid phase.
In some preferred embodiments, the monomer subunits of the invention are amino acids or their activated derivatives, protected by standard protecting groups known in the art. Preferred monomer subunits according to the present invention are amino acid compounds having formula (II): 
wherein:
L is a naturally-occurring nucleobase or a non-naturally-occurring nucleobase, or a protected derivative thereof;
R7xe2x80x2 is hydrogen or C1-C8 alkylamine;
E is COOH or an activated or protected derivative thereof; and
Z is NH2 or NHPg, wherein Pg is an amino-protecting group.
Preferably, R7xe2x80x2 is C3-C6 alkylamine. More preferably, R7xe2x80x2 is C4-C5, alkylamine. Still more preferably, R7xe2x80x2 is butyl amine.
The carbon atom to which substituent R7xe2x80x2 is attached (identified by an asterisks) is stereochemically enriched. Preferably, the stereochemical enrichment is of R configuration.
Preferably, compound (II) of the present invention is derived from an amino acid. More preferably, compound (11) of the present invention is derived from D-lysine.
One aspect of the present invention provides compounds of formula (III): 
wherein:
R8 is H or an amine protecting group;
R9 is H, alkyl having from 1 to about 12 carbon atoms, or alkenyl having from 2 to about 12 carbon atoms;
one of R4 and R5 is H and the other of R4 and R5 is a moiety of formula (IV) 
wherein
R10 is an amine protecting group;
Lxe2x80x2 is selected from the group consisting of hydrogen, hydroxy, (C1-C4)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, and heterocyclic moieties, reporter ligands, wherein amino groups are, optionally, protected by amino protecting groups;
A is a group of formula (IIa)-(IId): 
where:
X is O, S, Se, NR3, CH2 or C(CH3)2;
Y is a single bond, O, S or NR4 where R4 is as described above;
each r and s is zero or an integer from 1 to 5;
each R1 and R2 is independently selected from the group consisting of hydrogen, (C1-C4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and
R3 is selected from the group consisting of hydrogen, (C1-C4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C1-C4)alkyl, hydroxy, alkoxy, alkylthio and amino.
The present invention also provides a pharmaceutical composition comprising peptide nucleic acids of the present invention and at least one pharmaceutically effective carrier, binder, thickener, diluent, buffer, preservative, or surface active agent.
In other aspects, methods are provided for method for enhancing the DNA or RNA sequence specificity of a peptide nucleic acid by incorporating one pr more 2,6-diaminopurine nucleobases into the peptide nucleic acid backbone.